Co-expression of colligin and collagen in oral submucous fibrosis: plausible role in pathogenesis.

The high incidence of oral submucous fibrosis (OSF), a potentially malignant condition of the oral cavity, in the Indian subcontinent is causally associated with commonly prevailing habit of chewing areca nut and tobacco. Knowledge of molecular alterations in OSF is meagre. OSF is characterised by progressive accumulation of collagen fibres in lamina propria and oral submucosa. Colligin/HSP47 is a 47KDa stress protein which acts as a chaperone for collagen. We hypothesized that since colligin plays a vital role in folding and assembling collagen it may be involved in the pathogenesis of OSF. The present study was undertaken in tobacco and areca nut chewing Indian OSF patients to investigate the correlation, if any, between the expression of colligin and collagen type I proteins in OSF lesions. Immunohistochemical analysis showed overexpression of colligin and collagen type I proteins in 16/23 (70%) and 15/23 (65%) of OSF cases, respectively. The hallmark of the study was the significant association between the increased expression of type I collagen and its chaperone, colligin, in OSF lesions (P=0.0494). The data suggest that the increased levels of colligin in OSF may contribute to the deposition of collagen and consequent increased fibrosis in the oral submucosa in OSF lesions.

Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.

Interaction of procollagen I and other collagens with colligin.

Colligin is a collagen-binding glycoprotein of molecular mass 46000 Da localized to the endoplasmic reticulum (ER) of diverse kinds of cells that produce collagen I. In order to help define its role in collagen biosynthesis and to study the interaction of colligin with procollagen I in detail, the binding characteristics of colligin purified from L6 myoblasts have been studied. A total of 3 mol were found to bind/mol of procollagen I, with a Kd of about 25 nM. Both pure and separated pro alpha 1(I) and procollagen alpha 2 (I) chains were able to compete with procollagen I for binding to colligin. However, colligin binds to pro alpha 2 (I) with higher affinity than to pro alpha 1 (I). To find if the binding activity of colligin was altered during purification, an assay to measure colligin binding to procollagen in crude myoblast cell extracts was developed. This procedure gave the same binding parameters as did the highly purified colligin. Among different collagen types, colligin was found to bind to collagen I and collagen IV, but not to collagen III. In order to examine whether glycosylation or phosphorylation of colligin were required for the binding of colligin to procollagen I and to obtain enough colligin for further studies, recombinant protein was produced in Escherichia coli. An immunoaffinity purification scheme was used to get virtually pure protein in milligram yields. Comparison of the recombinant colligin with that isolated from L6 myoblasts showed that both types existed in solution as monomers and dimers. In addition, both types of colligins showed identical properties with regard to their binding to procollagen I and the isolated pro alpha 1(I) and pro alpha 2(I) chains. Post-translational modifications of colligin were thus not essential for binding to procollagen I.

Inhibition of procollagen I degradation by colligin: a collagen-binding serpin.

Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment.

A novel very high molecular mass protein located in the membrane skeleton.

A monoclonal antibody generated from a mouse immunized with L6 rat myoblast cells was found to react with a major 700-kDa band and a minor 500-kDa band in immunoblots. Immunofluorescence microscopy demonstrated a submembranous location in tissue sections and an exclusion from stress fiber regions in cultured cells. Further, permeabilization of cultured cells with nonionic detergent prior to fixation changed the diffuse pattern of fluorescence to a web. These findings are characteristic of membrane skeletal proteins. In muscle tissue, the protein was much more abundant in fast twitch fibers and was found in internal locations as well as at the membrane. The protein could be solubilized in the absence of detergents and, hence, is not transmembrane. Although initially discovered in myoblast cells, the protein is present in a variety of tissue types, including brain, kidney, heart, liver, and lung. Pulse-chase labeling of the two bands suggested that the 500-kDa band was not a breakdown product of the 700-kDa protein. The protein appears to be a previously undiscovered membrane skeletal constituent for which the name "endossin" is proposed.

Protein kinase A regulation of cAMP phosphodiesterase expression in rat skeletal myoblasts.

We have been studying cAMP signaling in L6 myoblasts because of its potential role in regulating the differentiation of these cells into multinucleate myotubes. Previous studies have shown that treatment of L6 myoblasts with cAMP analogs causes an increase in cAMP phosphodiesterase activity. To assess the role of protein kinase A in this cAMP-mediated increase in cAMP phosphodiesterase activity, L6 myoblasts were transfected with a plasmid containing the cDNA for a mutant regulatory subunit of protein kinase A, which functions as a dominant negative inhibitor of this enzyme. The cDNA was under control of the metallothionein promoter in the construct. Induction of the mutant regulatory subunit with Zn2+ decreased cAMP-dependent protein kinase activity by 90%. Zn2+ treatment was also able to completely block the cAMP-mediated increase in phosphodiesterase activity, showing that this effect is mediated by protein kinase A. The activity of the cAMP-induced phosphodiesterase was inhibited by low concentrations of RO 20-1724, showing that it was a member of the type IV low Km cAMP phosphodiesterase family of enzymes. We used the polymerase chain reaction and consensus primers designed to amplify phosphodiesterase sequences to show that L6 myoblasts also contain mRNA for a type IV low Km cAMP phosphodiesterase designated PDE3.1. The levels of this mRNA were increased greatly by treatment with dibutyryl cAMP or forskolin in L6 myoblasts and also in differentiated L6 myotubes. Run-off transcription assays showed that this increase in PDE mRNA was regulated, at least in part, by an increase in the rate of transcription of the PDE3 gene. The induction of PDE3 message by cAMP was blocked when the L6 transfectants were treated with Zn2+ to induce protein kinase A inhibition. Therefore, some of the cAMP-mediated increase in phosphodiesterase activity seen in L6 myoblasts is due to a protein kinase A-mediated increase in PDE3 mRNA. This pathway may serve as a feedback mechanism to modulate the inhibitory effects of cAMP on myogenesis.

Parallel regulation of procollagen I and colligin, a collagen-binding protein and a member of the serine protease inhibitor family.

A potential regulatory linkage between the biosynthesis of colligin, a collagen-binding protein of the ER, and procollagen I was examined under a variety of experimental conditions. Cell lines which did not produce a significant amount of procollagen I mRNA also lacked the capacity to produce colligin mRNA. Anchorage-dependent cell lines like L6 myoblasts and normal rat kidney fibroblasts produced both colligin and procollagen I mRNA, but the level of both was concurrently reduced considerably in their ras-transformed counterparts. Similarly, during the differentiation of L6 myoblasts, levels of both colligin and procollagen declined together. Treatment of myoblasts by dexamethasone or EGF led to a decrease in the steady-state levels of procollagen I mRNA, and this was, again, accompanied by a decrease in colligin mRNA synthesis. On the other hand, when the rate of procollagen I synthesis was stimulated by treatment of myoblasts with TGF beta, it led to the concurrent augmentation of both the mRNA and protein levels of colligin. A linkage between the regulation of synthesis of procollagen I and colligin thus seems to exist. The only exception to this generalization is provided by the heat induction behavior of the two proteins. Treatment of myoblasts for a very short period leads to an increase in the synthesis of both the mRNA and protein levels of colligin. This, however, is not accompanied by a change in the mRNA levels of procollagen I. These studies establish that colligin and procollagen are generally tightly co-regulated except after heat shock, suggesting an important functional linkage.

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.

Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins.

Several cDNA clones encoding a collagen-binding protein were isolated from human fibroblasts. The cDNA encoded a 417 amino acid protein, containing two potential N-linked oligosaccharide binding sites and a C-terminal RDEL sequence, which has been shown to act as an endoplasmic retention signal in other systems. The derived amino acid sequence of the protein shows close homology with gp46 from rat skeletal myoblasts, J6 protein from mouse F9 embryonal carcinoma cells and hsp47 from chick embryo fibroblasts. It also shows sequence similarity with members of the serpin family.

A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors.

Several cDNA clones encoding a 46-kDa collagen-binding glycoprotein (gp46) from rat skeletal myoblasts were isolated and sequenced. The cDNA encoded a 17-amino acid signal peptide and a 400-amino acid mature protein, containing three potential N-linked oligosaccharide attachment sites. The cDNA sequence of gp46 shows 93% identity in the coding region with J6, a retinoic acid-inducible gene coding for a protein of unknown function described from embryonal carcinoma F9 cells. The first 41 NH2-terminal amino acids of the predicted J6 sequence are, however, different from the gp46 sequence as a result of a 7-base pair insertion in the gp46 cDNA. In addition, the NH2-terminal amino acid sequence of hsp47, a collagen-binding protein found in chick embryo fibroblasts, shows 64% identity to gp46 over 36 residues. Interestingly, this alignment begins 10 residues inward from the first amino acid in the mature form of gp46. A significant sequence similarity was observed between gp46 and members of the serine protease inhibitor (serpin) family. Unlike other serpins, however, gp46 is both a heat shock and a collagen-binding protein and is localized to the lumen of the endoplasmic reticulum, as suggested by the presence of the RDEL sequence at the COOH terminus. This sequence is similar to other proposed endoplasmic reticulum retention signals.

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.

Two stress proteins of the endoplasmic reticulum bind denatured collagen.

A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42 degrees C, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification.

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen.

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts.

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.

Regulation of cyclic AMP-dependent protein kinase levels during skeletal myogenesis.

We showed previously that the levels of type I regulatory subunit of cyclic AMP-dependent protein kinase increase during differentiation of L6 skeletal myoblasts as a result of a specific decrease in its rate of degradation. Studies on the rates of degradation of the catalytic subunit show that unlike the type I regulatory subunit, catalytic subunit is degraded very slowly in myoblasts (t1/2 = 29 h) and more rapidly in myotubes (t1/2 = 14 h). As with the regulatory subunit, the degradation of catalytic subunit is increased by treatment of myoblasts with cyclic AMP analogues. These results suggest that the overall increase in the amount of type I cyclic AMP-dependent protein kinase holoenzyme during myogenesis is due to the increase in levels of mRNA for the catalytic subunit. This probably leads to an increase in the amount of catalytic subunit, which then stabilizes the regulatory subunit, thereby causing an increase in the levels of this protein also.

Glycoprotein glycans may not be necessary for the differentiation of skeletal myoblasts.

Previous work using glycosylation inhibitors has suggested that high-mannose type but not complex type oligosaccharides on the surface of cells may play a role in the differentiation of skeletal myoblasts. Earlier, we had shown that a concanavalin A-resistant mutant derived from an L6 myoblast line fails to differentiate in a medium containing 10% horse serum. Here we show that one such concanavalin A-resistant mutant (D-1) which was reported to have oligosaccharides of the type Man(3-5)G1cNAc2, shows significant fusion ability when grown in media containing 1% horse serum. Lowering the serum concentration did not alter the dolichol-phosphate mannosyltransferase activity in D-1 which remained at low levels compared to L6. The incorporation of [3H]mannose in D-1 was found to be 60% of L6 in 10% serum whereas in 1% serum the incorporation into D-1 was further reduced to 30% of L6. [3H]mannose-labeled ConA-binding proteins isolated from L6 were quantitatively and qualitatively similar in cells grown in either 10 or 1% serum. However, in D-1 cells a further decrease in the ConA-binding ability of these glycoproteins was observed. Biochemical differentiation also occurs in D-1 upon fusion in 1% serum as seen by the increase in mRNA levels of the muscle-specific markers myosin light chain and troponin T. These results suggest the high-mannose type of oligosaccharides may not be involved in myoblast differentiation.

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg-Gly-Asp sequence.

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.

Levels of type I cAMP-dependent protein kinase regulatory subunit are regulated by changes in turnover rate during skeletal myogenesis.

The regulation of type I cAMP-dependent protein kinase during the differentiation of L6 myoblasts has been investigated in order to assess a possible role for this enzyme in the control of myogenesis. Immunoblot analysis showed that the levels of the type I cAMP-dependent protein kinase regulatory subunit (RI) increased 3-fold during differentiation. However, measurement of RI mRNA levels using an RI cDNA probe showed that this increase was not regulated transcriptionally. Determinations of the turnover rate of RI demonstrated that the subunit becomes 3-fold more stable in the differentiated cells. Therefore, it appears that the increase is due to a decrease in the rate of degradation of RI. The increase in both the amount and stability of RI could be reversed by treating myotubes with cAMP analogues or forskolin. Furthermore, during differentiation there was a large decrease in cAMP levels. It was, therefore, concluded that the increase in RI levels seen during differentiation is probably due to an increase in the stability of the subunit as a result of a decrease in intracellular cAMP concentrations.

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.